Bacterial enterotoxins are members of a class of proteins known as superantigens (SAgs) that have lethal activity because they elicit massive T cell activation, leading to release of inflammatory molecules such as TNF-alpha. Most of the known bacterial SAgs are expressed by Staphylococcus aureus and Streptococcus pyogenes. These include select agents of bioterrorism such as staphylococcal enterotoxin B (SEB) that has been considered by the U.S. military as a major threat for incapacitation and lethality. SAgs activate T cells by cross-linking T cell receptor Vbeta regions with class II MHC molecules on another cell. The proposed project will develop soluble Vbeta receptors that can neutralize enterotoxins and will involve the laboratories of four scientists that have extensive experience in structural, functional, and clinical studies of SAgs: David Kranz, University of Illinois; Patrick Schlievert, U. Minnesota; Roy Mariuzza, U. Maryland; Sina Bavari, U.S. Army. The receptor engineering components of the project will use yeast display technology to generate high-affinity antagonists against a panel of SAgs (in the order of priority: SEB, TSST-1, SEC3, SpeA, SpeC). Based on the low LD50 values (approximately 1 mu g) of SEB predicted for humans, it is very likely that low picomolar affinity agents will be required for effective neutralization. In previous work, we snowed that high-affinity, soluble receptor domains (12 KDa) engineered by yeast display inhibited the in vitro activity of SEB and SEC3. As evidence that Vbeta:SAg interactions can be engineered to low picomolar affinities, we recently generated a panel of Vbeta8 mutants with KD values for SEB from 40 to 90 pM. The present project will further develop these, and other soluble Va receptors, as therapeutics. The specific aims are to: 1) To engineer and characterize Vbeta regions that bind with high-affinity to SAgs: SEB, TSST-1, SEC3, SpeA, and SpeC, 2) To generate Vbeta immunoglobulin fusions of the agents from aim 1 and to characterize their binding properties and serum lifetimes in mice, 3) To test the ability of soluble Vbeta-Ig fusions to inhibit activity of SAgs in vitro and to neutralize the toxins in animal models. The models will include "humanized" class II MHC/CD4 transgenic mice (including an aerosol model of exposure) and rabbits, which resemble human SAg-mediated diseases in symptoms and toxicity. The strategies developed here to engineer very high-affinity neutralizing receptors and to increase their serum lifetimes should be directly translatable to the development of antagonists against other toxins that are considered potential agents of bioterrorism.